Virus-specific T cells capable of controlling hepatitis B virus (HBV) and eliminating hepatocellular carcinoma (HCC) expressing HBV antigen (Ag) are deleted or dysfunctional in patients with chronic HBV infection. Current antiviral therapy targets the virus reverse transcriptase and rarely establishes immunological control over HBV replication. Adoptive cell transfer (ACT) of HBV-specific CD8+ cytotoxic T lymphocytes (CTL) is an highly promising treatment for chronic HBV infection and HBV-associated HCC. Naive or central memory T cell-derived effector CTL - the right or highly reactive CTL are the optimal populations for ACT-based immunotherapy, because these cells have a high proliferative potential, are less prone to apoptosis than terminally differentiated cells and have a higher ability to respond to homeostatic cytokines. However, such ACT is often not feasible due to difficulties in obtaining sufficient numbers of highly reactive CTL from patients. The long-term goal of our research program is to develop and optimize strategies for utilizing pluripotent stem cells (PSC) expressing Ag-specific T cell receptor (TCR) as a source of highly reactive CTL for cell-based therapies. The objective in this application is to determine the fundamental properties of HBV-specific CTL from induced PSC (i.e., iPSC-CTL) genetically modified with HBV-specific TCR, for potential immunotherapeutic development against chronic HBV infection and HBV-associated HCC. The central hypothesis is that HBV-specific iPSC-CTL developed by our in vitro differentiation system are naive monoclonal HBV-specific CD8+ T cells, which can be used to generate highly reactive HBV-specific CTL for cell-based therapies. Guided by published results and preliminary data from the applicant's laboratory, this hypothesis will be tested by pursuing two specific aims: 1) to define the fundamental properties of HBV-specific iPSC-CTL that relate to their anti-HBV activity; and 2) to determine the anti-HBV activity of HBV-specific iPSC- CTL in HBV-bearing mice. Under the first aim, in vitro and in vivo approaches, which have been established as feasible in the applicant's laboratory, will be used. The fundamental properties of HBV-specific iPSC-CTL regarding phenotype, specificity and function will be defined. Under the second aim, using murine models of HBV infection and HBV-associated tumor, the efficacy of HBV-specific iPSC-CTL for cell-based therapies will be determined. The approach is innovative, because HBV-specific iPSC-CTL genetically modified with HBV- specific TCR for cell-based therapies of chronic HBV infection and HBV-associated HCC has not been previously explored. The proposed research is significant, because it will provide new insight and strategies for generating highly reactive virus-specific PSC-T cells and in so doing drive forward use of therapeutic T cells for the treatments of infectious diseases and cancers.